The nervous system comprises an extensive array of neuronal pathways that control and modulate virtually all bodily functions. This system develops from a simple neuroectoderma tube, and through a series of processes regulating cell division, commitment, migration, and differentiation, forms functional synaptic connections in a highly specific fashion. These processes involve internal cell programs controlling stage-specific gene expression, cell-cell interactions, and chemical signalling. The result is an organ system of remarkable complexity, responsible for the full range of physiological and intellectual behavior of higher organisms. A major challenge to neurobiologists is understanding the specific cellular and molecular interactions that mediate the formation and/or maintenance of functional connections between particular subsets of neurons and their target cells (Purves, 1988). This goal is made exceedingly difficult by the extensive heterogeneity of cell types and connections even within specific brain regions. For these reasons, a variety of primary cell culture techniques have been developed to provide an approach to simplification of the system for experimental purposes.
These techniques are still limited by the cell heterogeneity of specific regions of brain and the difficulty of obtaining sufficient numbers of cells of a specific type for detailed biochemical and molecular biological studies. One approach that has been employed to circumvent these problems has been the utilization of clonal cell lines that exhibit neuronal features (Bottenstein 1981; Schubert 1984; Banker and Goslin 1991). For example, PC12 cells, arising from a rat pheochromocytoma, have been used in a variety of studies to elucidate the response to and the mechanism of action of nerve growth factor (NGF) (Tishler, et al. 1975; Greene, et al. 1991). Although this approach has provided a considerable amount of information, it is limited for the following reasons.
First, these cell lines are derived from spontaneously arising tumors and therefore carry with them the inherently malignant nature of their cells of origin. Although a recent report has described the isolation of a human neural cell line from nonneoplastic tissue (Ronnett, et al. 1990), the frequency of such spontaneous `immortalization` events from primary neuronal cells is extremely low. Therefore, the possibility of obtaining cell lines of a specific neurochemical type is unlikely. Second, most of the cell lines currently available represent subclones of single tumors, arising most frequently in the peripheral nervous system, i.e. pheochromocytomas and neuroblastomas, and are therefore of limited usefulness for studying specific processes within particular CNS pathways.
Relatively little work has been carried out with respect to the establishment of permanent cell lines from specific brain regions that elaborate or respond to trophic signals which are involved in the establishment and maintenance of the synaptic circuitry of those regions. Two general strategies are available for engineering such cell lines.
The first is the use of retroviral-mediated introduction (transduction) of oncogenes to `immortalize` primary brain cells. While this approach is useful for studying the properties of progenitor cells, it is of more limited utility as an approach to the study of differentiated cells. Retroviral transduction is only effective with mitotic cells since the retroviral DNA can only be inserted into the host genome during replication. In addition, once a cell is `immortalized`, it tends to remain locked within a particular developmental window, and in fact, this phenomenon has been exploited by immunologists to study the stages of lymphocyte differentiation (Paige, et al. 1989; Alt, et al. 1987). Therefore, while viral gene transduction might yield cell lines for the study of early stages of neuronal development, it is less likely to provide cell lines that express the phenotypic repertoire of mature neurons which are almost invariably post-mitotic.
A second approach has employed somatic cell fusion techniques in which primary brain cells are fused to a neuroblastoma cell line by exposure to polyethylene glycol (Hammond, et al. 1986). The fusion technique allows one to `immortalize` cell populations that are post-mitotic and therefore more likely to express highly differentiated neuronal phenotypes. While no current `cell immortalization` strategy is devoid of limitations, the somatic cell fusion technique makes available large numbers of brain-region-specific clonal cells for cellular and molecular studies of specific neural circuits.
The technique of somatic cell fusion has been widely used to study a variety of cellular and genetic questions (Shay, 1982). Perhaps one of most noteworthy applications has been the generation of lymphoid hybridoma cell lines to produce monoclonal antibodies (Kohler and Milstein, 1975). In the nervous system, somatic cell fusion has been applied to the development of several cell lines derived from sympathetic neurons (Greene, et al., 1975) or dorsal root ganglion cells (Platika, et al., 1985). The cell fusion approach has been previously exploited because of its potential for immortalizing central neurons that are post-mitotic and therefore committed to a particular neurochemical phenotype as well as neuroanatomical pathway (Hammond, et al. 1986; Hammond, et al. 1990; Lee, et al. 1990).